I have a list of questions for you guys to think and we will discuss tomorrow. You guys should be able to answer these questions with a little more effort!
Miniprep Handbook
C:\Documents and Settings\Jingwei_Zhang\My Documents\My Dropbox\Undergraduate Research Training\Crash Course\Basic Wet Lab Protocols\QIAprep_Miniprep_Handbook.pdf
Go to p.39 Table: origin of replication and copy number, one of the major factors that affect DNA yield!
p. 40: What is DNA methylation? What does it do?
What is par locus? What does it do? How does it affect plasmid segragation? What implication does it have with regard to adding antiobiotics to maintain plasmids?
Amp is very sensitive to temperature, what does it say about preparing Amp Agar Plate? Can we add Amp directly to the heated LBAgar media? What advantage does Carbenicillin have over Amp?
p. 41, concentration of normally made antibiotics!
What is LB media?
Gel Purification:
I went through Gel Extraction. Think about these questions and understand them for the Gel Extraction Procedure!
You can find the answer in C:\Documents and Settings\Jingwei_Zhang\My Documents\My Dropbox\Undergraduate Research Training\Crash Course\Basic Wet Lab Protocols\QIAGEN_QIAquickSpin_EN.pdf
Take a look at the diagram on p.10, understand the DNA size range of purification.
What is the component in the QG buffer, the PE buffer and the EB buffer? Why can we bind DNA using QG buffer while eluting with EB buffer?
Check out:Preparative and analytical purification of DNA from agarose; PNAS-1979-Vogelstein, the article is in the same protocols folder.
During DNA miniprep, sometimes people elute DNA with TE buffer. What is TE buffer? Why it is not recommended that we use them for Gel Purification?
p.14: Take a look at the relationship between elution volume and extraction efficiency. Also, look at the impact of incubating one minute before spin eluting DNA.
p.15, how do we quantify DNA?
p.17: Diagram of our Purify procedure, understand the process.
p. 23-24: QIAquick Gel Extraction Kit Protocol
What's the capacity of the column in each tube? If we are eluting DNA with 50uL EB buffer and ended up with ~ 45uL, what is the maximum DNA concentration we can get?
What is isopropanol used for? What DNA size range is this step useful?
Why is step11 important? (got an answer? what will Ethanol do if it remains there!)
What is the impact of letting the column sit for 1min?
What is the elution volume that we use? (REMEMBER! We start everything with digesting 500ng of plasmid in a total 20uL reaction volumn. We ran a gel and we are purifying from the gel product) Ligation: Check out for the T4 ligase enzyme and buffer backgrounds: http://jingwei-labtraining.wikispaces.com/BWL
What is included in T4 ligase buffer? What is the component of the T4 ligase enzyme? Why can we not add too much enzyme?What is the role of DTT? What is the role of ATP? Why not use NAD+ instead of ATP? Is there a difference if we are ligating an overhang DNA pieces vs. blunt end DNA pieces?
Note! go further down to the FAQ at the bottom half of the page!
Q1: read carefully the common trouble shooting. We utilized CIP phosphatase to remove the phosphate in our digestion vector, yet as the A1 mentioned, CIP phosphatase will interfere with ligation reaction, so when is the CIP protein removed during our experiment? What's the impact of using PEG? Is the ligation mixture suitable for electroporation into e- competent cells, what can you do to make the reaction mixture suitable for electroporation?
Q2:
Q3:
Q7: Can T4 ligase be heat inactivated?
Q9: why is 1:5 the nice ratio? what will happen if we use 1:1? what will happen if we use 1:6? What does multiple inserts mean?
Where does all the volume calculation in Satoshi's protocol come from?
What is the total amount mass of vector DNA we are adding? What is our assumption? How do we calculate the insert volume and how do we calculate the water volume? According the gel I sent you guys, does this calculation hold?
Why is it important to spin down the liquid?
What is room temperature? Transformation:
Science Principle (\Dropbox\Undergraduate Research Training\Crash Course\Basic Wet Lab Protocols\Summary of science behind the DNA plasmid Miniprep Kit.docx)
), Protocol, check out the Qiagen Miniprep Handbook.pdf (My Dropbox\Undergraduate Research Training\Crash Course\Basic Wet Lab Protocols), Qiagen Buffers,
Agarose gel preparation for DNA seperation and gel electrophoresis: background
Agarose
SYBR® Safe DNA Gel Stain: SYBR Green I(learn the science, but note the safety section as well), FAQ(check out the imaging section)
Ethidium Bromide, how people used to image DNA on gel?
GeneRuler 1kb+ DNA ladder: Product info, 0.5ug/uL
DNA ligation using T4 Ligase:
T4 DNA Ligase and T4 Ligase Buffer Product Description
Ligation Protocol
Western Blot Hub
Genscript Protocol (Dropbox\Undergraduate Research Training\Crash Course\Basic Wet Lab Protocols)
Comparison between PVDF and nitrocellulose membrane
I have a list of questions for you guys to think and we will discuss tomorrow. You guys should be able to answer these questions with a little more effort!
Miniprep Handbook
C:\Documents and Settings\Jingwei_Zhang\My Documents\My Dropbox\Undergraduate Research Training\Crash Course\Basic Wet Lab Protocols\QIAprep_Miniprep_Handbook.pdf
What are the formulas for Qiagen Miniprep?
P1, P2, N3, PE, EB?
http://openwetware.org/wiki/Qiagen_Buffers
Go through all the yellow notes.
There is very great background information contained in Appendix A.
Go to p.36 for troubleshooting
Go to p.39 Table: origin of replication and copy number, one of the major factors that affect DNA yield!
p. 40: What is DNA methylation? What does it do?
What is par locus? What does it do? How does it affect plasmid segragation? What implication does it have with regard to adding antiobiotics to maintain plasmids?
Amp is very sensitive to temperature, what does it say about preparing Amp Agar Plate? Can we add Amp directly to the heated LBAgar media? What advantage does Carbenicillin have over Amp?
p. 41, concentration of normally made antibiotics!
What is LB media?
Gel Purification:
I went through Gel Extraction. Think about these questions and understand them for the Gel Extraction Procedure!
You can find the answer in C:\Documents and Settings\Jingwei_Zhang\My Documents\My Dropbox\Undergraduate Research Training\Crash Course\Basic Wet Lab Protocols\QIAGEN_QIAquickSpin_EN.pdf
Take a look at the diagram on p.10, understand the DNA size range of purification.
What color is not appropriate when dissolving a gel? What does that mean? Why PH is so important?
http://en.wikipedia.org/wiki/DNA_separation_by_silica_adsorption
What is the component in the QG buffer, the PE buffer and the EB buffer? Why can we bind DNA using QG buffer while eluting with EB buffer?
Check out:Preparative and analytical purification of DNA from agarose; PNAS-1979-Vogelstein, the article is in the same protocols folder.
During DNA miniprep, sometimes people elute DNA with TE buffer. What is TE buffer? Why it is not recommended that we use them for Gel Purification?
p.14: Take a look at the relationship between elution volume and extraction efficiency. Also, look at the impact of incubating one minute before spin eluting DNA.
p.15, how do we quantify DNA?
p.17: Diagram of our Purify procedure, understand the process.
p. 23-24: QIAquick Gel Extraction Kit Protocol
What's the capacity of the column in each tube? If we are eluting DNA with 50uL EB buffer and ended up with ~ 45uL, what is the maximum DNA concentration we can get?
What is isopropanol used for? What DNA size range is this step useful?
Why is step11 important? (got an answer? what will Ethanol do if it remains there!)
What is the impact of letting the column sit for 1min?
What is the elution volume that we use? (REMEMBER! We start everything with digesting 500ng of plasmid in a total 20uL reaction volumn. We ran a gel and we are purifying from the gel product)
Ligation:
Check out for the T4 ligase enzyme and buffer backgrounds: http://jingwei-labtraining.wikispaces.com/BWL
What is included in T4 ligase buffer? What is the component of the T4 ligase enzyme? Why can we not add too much enzyme?What is the role of DTT? What is the role of ATP? Why not use NAD+ instead of ATP? Is there a difference if we are ligating an overhang DNA pieces vs. blunt end DNA pieces?
Note! go further down to the FAQ at the bottom half of the page!
Q1: read carefully the common trouble shooting. We utilized CIP phosphatase to remove the phosphate in our digestion vector, yet as the A1 mentioned, CIP phosphatase will interfere with ligation reaction, so when is the CIP protein removed during our experiment? What's the impact of using PEG? Is the ligation mixture suitable for electroporation into e- competent cells, what can you do to make the reaction mixture suitable for electroporation?
Q2:
Q3:
Q7: Can T4 ligase be heat inactivated?
Q9: why is 1:5 the nice ratio? what will happen if we use 1:1? what will happen if we use 1:6? What does multiple inserts mean?
Where does all the volume calculation in Satoshi's protocol come from?
What is the total amount mass of vector DNA we are adding? What is our assumption? How do we calculate the insert volume and how do we calculate the water volume? According the gel I sent you guys, does this calculation hold?
Why is it important to spin down the liquid?
What is room temperature?
Transformation:
What is DH10B? Why is it useful for transforming plasmids? Can we use it for expressing protein in pET28b vector?
Check out E. coli genotype: http://openwetware.org/wiki/E._coli_genotypes
Find the difference between DH10B, and BL21(DE3).
What is the total amount of DNA in ligation mixture, what is the theoretical amount of ligation plasmids in this mixture?
What is 5XKCM solution? Why is it useful for promoting DNA transformation efficiency?
What does heat shock do? and why do we need to follow by putting the heat shocked bacteria on ice? http://www.ncbi.nlm.nih.gov/pubmed/18651316?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=4&log$=relatedarticles&logdbfrom=pubmed
We are culturing for ~ 40-60min. What will happen if we over incubated the cells? Will we get more colonies or less colonies?